Open-Source Multiparametric Optocardiography

Since the 1970s fluorescence imaging has become a leading tool in the discovery of mechanisms of cardiac function and arrhythmias. Gradual improvements in fluorescent probes and multi-camera technology have increased the power of optical mapping and made a major impact on the field of cardiac electrophysiology. Tandem-lens optical mapping systems facilitated simultaneous recording of multiple parameters characterizing cardiac function. However, high cost and technological complexity restricted its proliferation to the wider biological community. We present here, an open-source solution for multiple-camera tandem-lens optical systems for multiparametric mapping of transmembrane potential, intracellular calcium dynamics and other parameters in intact mouse hearts and in rat heart slices. This 3D-printable hardware and Matlab-based RHYTHM 1.2 analysis software are distributed under an MIT open-source license. Rapid prototyping permits the development of inexpensive, customized systems with broad functionality, allowing wider application of this technology outside biomedical engineering laboratories.


SUPPLEMENTARY TEXT
Lab Jack Each lab jack was assembled using the 6-step method illustrated in Figure 2a. Once assembled, supported components are secured using the assembly mechanism illustrated in Supplementary Figure 1. The weight limit of the lab jack is 30kg.
Lab Jack Assembly Instructions: In step I., the left and right supports (perfusion or optical) were secured to the plate hinge with nuts (Supplementary Figure 2).
Step I. was repeated three times to complete four sets of plate hinges with supports attached. In step II., one side of the lab jack was assembled first, with one plate hinge attached to the underside of the top plate and a second plate hinge to the bottom plate on the same side. The plate hinges were secured using lab jack twist locks. In step III., the mid-hinges (one threaded and one un-threaded) were secured to the supports with nuts. In step IV., the remaining two plate hinges were attached to the top and bottom lab jack plates. The other side of the mid-hinges were then secured to the supports with nuts. While at its lowest height and with the top and bottom plates parallel to each other, the screw was placed through the mid-hinges with the distal end going through the un-threaded mid-hinge first (step V.), reaching its correct position with the screw head positioned flat against the un-threaded midhinge (step VI.). Finally, the bottom plate of the lab jack was secured to the breadboard using M6 screws and washers.

Tilting Platform
The tilting platform (Figure 2b) was used to switch the optical components on the optical lab jack from sideways to upright imaging mode.
Tilting Platform Assembly Instructions: First, the platform raise was attached to the optical lab jack top plate using the sliding rails. The platform raise was secured in the horizontal direction using stoppers (Supplementary Figure 3). Next, the plate mount was attached to the platform raise using sliding rails and positioned flush with the front edge of the platform raise. The upright plate was then attached to the plate mount using an axial rod (Supplementary Figure 4). With the upright plate at rest in the horizontal position, the excitation and emission filter cubes were secured onto the upright plate using the sliding rails and the filter cube twist locks. For sideways imaging, the upright plate was left at rest in the horizontal position. For upright imaging, the upright plate was rotated 90° to the upright position and held in place using the upright stabilizer, which slides onto the slots on the plate mount and the underside of the upright plate. A third stopper was placed at the edge of the upright plate closest to the rotational axis for further support of the optical components.

Hydraulic Lift
The hydraulic lifts were used to vertically support and spatially align the cameras in both orientations. Two 25-35cm range hydraulic lifts supported the cameras during sideways imaging, while one 44-54cm height range supported the second camera during upright imaging. The weight limit of the hydraulic lift in 15kg.
Hydraulic Lift Assembly Instructions: The components of each hydraulic lift ( Figure 2c) include two 60mL syringes (Cat# 13-689-8, Fisher Scientific), a short length of silicon tubing to fit the syringes (Item # EW-96400-14, Cole-Parmer), a stopcock (Cat# 120722, Radnoti), and the 3D-printed components. To assemble, first, the 3D-printed base was attached to the breadboard with M6 screws. Next, a short piece of tubing was attached to an empty syringe that was then placed into the 3D-printed base with the tubing going through a hole centered at the bottom of the 3D-printed base. The 3D-printed top was then glued with a 1:1 epoxy-resin mixture to the handle of this syringe. Next, the second syringe was filled with 60mL of water, attached to a stopcock, and then secured to the free end of the silicon tubing using the stopcock. This syringe was used to adjust the height of the hydraulic lift (Supplementary Figure 5). At the desired height, the stopcock was placed in the off position.
Focusing the Cameras to Infinity The cameras were focused to infinity prior to signal alignment. With the optical components fully assembled, the projection lens sleeves were detached from the emission filter cube, leaving everything else intact. By rotating each focal adjuster to adjust the distance between each camera and its lens, the cameras were focused onto a distant object. Once each camera was focused onto the distant object, the detached components were re-attached to the emission filter cube using the projection lens sleeves. We recommend that the cameras be refocused each time the system is re-assembled.

Emission Filter Cube Dichroic Mirror Alignment
The dichroic mirror in the emission filter cube splits the voltage and calcium signals and directs the signals to the two cameras. To ensure spatial alignment (each camera has the same field of view) between the two cameras, the dichroic mirror angle and camera height was adjusted as needed before each experiment. This process should be performed after focusing each camera. We recommend that the dichroic mirror alignment be performed prior to each experiment for calibration and to ensure consistency between experiments.

Signal Alignment Instructions:
First, the MiCAM system was turned on and the MiCAM imaging software was opened. In the acquisition setup menu, the camera mode was set to 'Dual-Camera (Camera2: Horizontal mirror).' Next, the Focus Monitor was turned on. To align the image along one axis, the adjustable optics holder in which the dichroic mirror was placed was adjusted at the pivotal axis shown in blue in Figure 3. To align the image along the second axis, the hydraulic lifts were used to finely adjust the camera height. Once the cameras appeared to have the same field of view on the focus monitor display, the Dual Overlap function (Supplementary Figure 6, 4 th column) overlaid the two images to allow for any further adjustment. To secure the placement of the adjustable optics holder when satisfied with the alignment, two clips were placed on either side along the rail inside the emission filter cube. Finally, the top piece of the adjustable optics holder set that houses the emission filters was placed into the emission filter cube.
The subtraction of the two images from Camera 1 and Camera 2 (Supplementary Figure 6 column 3) was used to quantify image alignment. The images from each camera were converted to binary values to do a direct comparison. White pixels represent a mismatch between the two images while black pixels represent a match. By comparing the ratio of black pixels to the total number of pixels in the image, we demonstrate 99.07% alignment (aligned, top) and 85.62% (unaligned, bottom).

List of Components
Supplementary Tables 1-3 in the Excel document display and describe the individual parts in each component category: stage, optical, and perfusion. Each row in each table represents one CAD file that can be found on the open-source platform. Duplicates of parts are indicated in parenthesis next to the part name.

Cost Analysis
Supplementary Tables 4-6 in the Excel document present a cost breakdown and cost comparisons of 3D-printed components with commercially available parts for the full dual camera tandem lens system.  Table 1 of the manuscript.

Software Validation Supplementary
Additional Information: System Performance ABS Plus plastic is resistant to corrosion, sagging, aging, fraying and warping under the conditions of use. It is not recommended to use other plastics such as ABS or PLA because these are less resistant to mechanical stress. PLA also has a lower melting point than ABS and ABSP causing tissue chambers made out of PLA to warp when exposed to warm perfusate. Build-up of dried perfusate may cause breakage of the lab jack twist locks when under stress, so we recommend printing extra incase one needs to be replaced. While the ABS Plus plastic is not resistant to vibration, optical mapping experiments are best performed on an air table or steel breadboard that absorbs vibrations from the environment. The acetone vapor bath used to waterproof the tissue chambers will cause minimal warping of the plastic due to melting. This will cause the inlet and outlet holes for the superfusate to become slightly smaller in diameter, which is a consideration when modifying the chambers for different tubing sizes.
Additional Information: Modification of Parts Stage Components -Possible modifications include adjusting the lengths of the lab jack supports or the base component of the hydraulic lift to accommodate different height requirements. Lab jack and tilting platform plates can also be modified to secure different sized attachments. The camera cage dimensions can also be edited to fit a different sized camera that can still be attached to the camera end sleeve using the twist lock mechanism.
Optical Components -The mirror and filters holders can be modified to secure mirrors and filters of different sizes and shapes. This can be done by modifying the dimensions of the slots in the optics holders. The lens holders (objective and projection) can also be modified in inner diameter to fit different sized lenses.
Perfusion Components -The tissue chambers can be scaled up or down to fit a different sized preparation. Further, if additional fields of view are desired, additional slots can be added for the placement of more optical windows. For larger tissue preparations, it is important to take extra caution when waterproofing due to the additional pressure on the plastic surface that accompanies increased bath volume. It may be necessary to thicken walls of chambers for larger preparations. Table 7 Figure 1: Twist lock (a) and sliding rail (b) mechanisms permit attachment of parts throughout system.

Stage Components Parts List
Lab Jack

Platform Raise
Mounts directly onto Optical Lab Jack to provide option for upright imaging.

Upright Plate
Secures optical components.
Attached to the Plate Mount using the Axial Rod.

Upright Stabilizer
Secures Upright Plate to the Plate Mount to hold optical components at a right angle.

Upright Plate Mount
Mounts directly onto Platform Raise.

Axial Rod
Secures Upright Plate to Upright Plate Mount. Provides axis of rotation for Upright Plate.

Stopper (3)
Two are placed in slots on Optical Lab Jack on both ends of the Platform Raise. One is placed on the Tilting Platform in front of the Excitation Filter Cube when in upright imaging mode.

Upright Bath Lift (16-23cm)
Provides vertical adjustment of Upright Bath. A screw secures the position of the lift. The base piece (left) is screwed onto the breadboard.

Camera Cage (2)
Attaches camera to Camera End Sleeve.
For questions, please contact: optocardiography@gmail.com

Tilting Platform
Part Name/ Description

Objective Lens Sleeve Set
The objective lens is placed as shown in the photo. The cap is then screwed on to secure lens placement.

Objective Lens Sleeve Cover
Covers the objective lens when the system is not in use.

Excitation Filter Cube
Houses the Stationary Optics Holder. An excitation filter is placed in the small cylindrical slot (left).

Stationary Optics Holder
Holds dichroic mirror in rectangular slot. For single-camera studies, an emission filter is placed in the circular slot. The dichroic mirror guides excitation light to tissue preparation and splits emission light from excitation light.

Emission Filter Cube
Houses Adjustable Wall and Emission Filter Holder.

Adjustable Wall
Holds dichroic mirror that splits the voltage and calcium signals.

Emission Filter Holder
Holds emission filters that pass the wavelength of the emission spectra of each dye.

Optical Components Parts List
Placed on curved rail inside Emission Filter Cube to hold Adjustable Wall in place.

Filter Cube Twist Locks (4) and Tool
Secure Emission and Excitation Filter Cubes to Tilting Platform (or Optical Lab Jack).

Focal Adjuster (2)
Adjusts the focus of projection lenses to infinity.

Camera End Sleeve (2)
Attaches camera to Focal Adjuster.

Emission Filter Cube Cap
Placed on Projection Lens attachment site when doing single-camera studies.
For questions, please contact: optocardiography@gmail.com

Sideways Bath Linear Stage Mount
Mounts the Sideways Bath Stage to Perfusion Lab Jack.

Sideways bath Sliding Stage
Houses Sideways Bath and Cannula Holder. Placed onto Sideways Bath Linear Stage Mount.

Placed into Cannula Holder
Base. Permits vertical adjustment of cannula position.

Upright Bath
Houses tissue slice preparation or other preparations (e.g. atrial prep) for upright imaging.

Upright Bath Stage
Two screws secure the Cannula Holder Adjuster onto the Cannula Holder Rod and threeprong extension clamp. One screw secures the Cannula Holder Rod to the Cannula Holder Base.
Attaches to Cannula Holder Rod and three-prong extension clamp. Permits vertical and horizontal adjustment of cannula.

Cannula Holder Base
Placed directly into slot on Sideways Bath Stage.

Perfusion Components Parts List
Part Name/ Description Sideways Imaging

Sideways Bath and Screw
Houses whole-heart preparation for sideways imaging. Screw secures Electrode Paddle.

Electrode Paddle
Placed in Sideways Bath. Holds pseudo-ECG electrodes and stabilizes heart against optical window of bath.

Sideways Bath Stage
For questions, please contact: optocardiography@gmail.com

Upright Imaging
Houses Upright Bath and is mounted onto the Upright Bath Sliding Stage.

Stage Components
Note: Implementation costs were calculated based on the requirements of a dual-camera tandem lens system. The 3D-printed cost of implementation for each part is calculated in Table 5. Commercial cost of implementation for each part is calculated in Table 6. For commercial components with a range of cost options, as shown in